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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
3 September - 22 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Conducted according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Rhodium trinitrate
EC Number:
233-397-6
EC Name:
Rhodium trinitrate
Cas Number:
10139-58-9
Molecular formula:
HNO3.1/3Rh
IUPAC Name:
rhodium trinitrate
Constituent 2
Reference substance name:
Rhodium (III) nitrate
IUPAC Name:
Rhodium (III) nitrate
Test material form:
other: solution
Details on test material:
- Name of test material (as cited in study report): rhodium (III) nitrate solution
- Substance type: brown/red liquid
- Physical state: liquid
- Analytical purity: no data (assumed 100%)
- Impurities (identity and concentrations): metallic impurities: Ag, Al, As, Au, Ba, Bi, Co, In, Ir, K, Mg, Mn, P, Pb, Pd, Pt, Ru, Sb, Se, Sn, Sr, Te, all ND; Ca, 52 ppm; Cr, 63 ppm; Cu, 8 ppm; Fe, 230 ppm; Na, 675 ppm; Ni, 123 ppm; Si, 28 ppm; Zn, 8 ppm (wrt metal)
- Composition of test material, percentage of components: rhodium, 12.60% w/v; chloride (wrt Rh), 0.16% w/v
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: GB0143
- Expiration date of the lot/batch: 20 May 2017
- Stability under test conditions: no data
- Storage condition of test material: 15-25 deg C protected from light in a tightly closed container

Method

Target gene:
histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
mammalian liver post-mitochondrial fraction (S9) from male Sprague Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
Range-finder and Experiment 1: 5, 16, 50, 160, 500, 1600 and 5000 µg/plate
Experiment 2: 160, 300, 625, 1250, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: well-known solvent/vehicle
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Strain TA98 -S9 5 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100, TA1535 -S9 2 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537 -S9 50 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
TA102 -S9 0.2 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
TA98 +S9 10 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA100, TA1535, TA1537 +S9 5 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA102 +S9 20 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: Range-finder experiment (with and without S9), Experiment 1 (with and without S9) and Experiment 2 (without S9): in agar (plate incorporation); Experiment 2 (with S9): preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 3 days

DETERMINATION OF CYTOTOXICITY
- Method: The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.
Evaluation criteria:
Acceptance Criteria: The assay was considered to be valid if all the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges
2. The positive control chemicals induced increases in revertant numbers of ≥1.5 fold (in strain TA102), ≥2 fold (in strains TA98 and TA100) or ≥3 fold (in strains TA1535 and TA1537) the concurrent vehicle control confirming discrimination between different strains, and an active S9 preparation.
Evaluation Criteria: For valid data, the test article was considered to be mutagenic if:
3. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control values
4. The positive trend/effects described above were reproducible.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if [n]either of the above criteria were met.
Statistics:
None

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
in Experiments 1 and 2
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 and 5000 µg/plate, with and without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
in Experiments 1 and 2
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate, with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
in Experiments 1 and 2
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 and 5000 µg/plate, without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in Experiments 1 and 2
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate, with and without S9, plate incorporation; at 1250, 2500 and 5000 µg/plate, with S9, pre-incubation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in Experiments 1 and 2
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate, with and without S9, plate incorporation; at 1250, 2500 and 5000 µg/plate, with S9, pre-incubation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
in Experiment 1, with and without S9; in Experiment 2, with S9; also just below +ve threshold in Experiment 2, without S9
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate, with and without S9
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Range-finder experiment: stock solution, 50 mg rhodium (III) nitrate solution/ml, pH 1.86; Experiments 1 and 2: stock solution, 50 mg rhodium (III) nitrate solution/ml, pH 1.63. "There is no evidence that low pH causes mutations in the Ames test" (Kirkland DJ (2012). Review of the effect of pH on the integrity of in vitro and in vivo genotoxicity assays. Report prepared for The Precious Metals & Rhenium Consortium).
- Precipitation: at 2500 and 5000 µg/plate, pre-incubation (Experiment 2, with S9, TA1535 and TA1537)

RANGE-FINDER EXPERIMENT: strains TA98, TA100 and TA102; evidence of toxicity in the form of a slight thinning of the background bacterial lawn was observed at 5000 µg/plate in all strains in the absence of S9

COMPARISON WITH HISTORICAL CONTROL DATA: mean vehicle control counts were comparable with the laboratory’s historical ranges; mean positive control counts were generally within or higher than the laboratory's historical control ranges (except TA102, experiment 1, without S9: 528 (historical control range 568-1290)
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
In a guideline study, to GLP, rhodium (III) nitrate solution induced mutations in histidine-requiring strains TA98 and TA102 of Salmonella typhimurium when tested up to precipitating concentrations in the absence and presence of a rat liver metabolic activation system (S9), and in strain TA100 in the presence of S9 alone.
Executive summary:

Rhodium trinitrate solution (12.9% rhodium) was tested in a bacterial reverse mutation (Ames) assay, conducted according to OECD Test Guideline 471 and to GLP. The test substance was assayed at test concentrations of up to 5000 µg/plate (it is unclear whether this relates to rhodium nitrate itself or to rhodium nitrate solution), in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of S. typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S9). Two experiments were conducted (each in triplicate): initially a plate incorporation protocol was used; the experiment was repeated using a pre-incubation protocol for those strains that were negative in the first experiment.

There was some evidence of toxicity at the highest concentration tested with the plate incorporation protocol and at 1250 -5000 µg/plate with the pre-incubation protocol. Vehicle and positive controls performed as expected. Strain TA98 was positive for mutagenicity in both experiments, with and without S9. Strain TA100 was positive for mutagenicity in both experiments with S9 alone. Strain TA102 was positive in both experiments with S9, in the first experiment without S9 and approaching the threshold for a positive response in the second experiment without S9. Strains TA1535 and TA1537 were negative in both experiments, with and without S9.

Under the conditions of this study rhodium (III) nitrate solution was clearly mutagenic.